PILOT PROJECT 8: HUMORAL IMMUNITY TO A NOVEL PNEUMOCYSTIS CARINII ANTIGEN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 1)重组蛋白质的质粒构建和表达。为了比较我们新的p55蛋白（p55-v3）与先前描述的p55蛋白（p55-v0）的抗原性，我们从辛辛那提大学的Smulian博士处获得了p55-v0质粒构建体。出乎意料的是，我们发现该构建体不表达重组蛋白。多个克隆的DNA序列分析显示编码区中间的单个核苷酸A缺失，导致阅读框改变和下游多个终止密码子的引入。因此，我们自己构建了新的构建体，其中一个构建体用于全长p55-v0序列，另外一个构建体用于三个重叠片段，其对应于我们之前表达的p55-v3片段。 2)肽的合成和抗血清的产生：我们已经从Sigma获得了合成肽和抗血清，一种对p55-v0特异，两种对p55-v3特异。抗血清将用于研究特定目标1中提出的p55-v3和p55-v0的表位定位和差异表达模式。 3)动物模型：40只大鼠分为3组。A组（10只大鼠）为对照组，饲养在SPF条件下，不接受任何药物治疗。将B组（20只大鼠）和C组（10只大鼠）共同饲养在常规房间（无过滤器顶部的开放笼）中。B组给予Depo-medrol免疫抑制，诱发自发性卡氏肺孢子虫肺炎（PCP）。使用的Depo-Medrol剂量为2 mg/100 g体重，前4周每周两次皮下注射，然后每周一次，持续6周。C组未未接受免疫抑制，但预期自然暴露于B组卡氏肺孢子虫。在免疫抑制3-4周后，B组中的所有大鼠都生病并且具有明显的体重减轻。5只大鼠在免疫抑制后8-10周死亡。通过染色和PCR检查这5只大鼠的肺，发现它们中没有卡氏肺孢子虫阳性。此外，我们用卡氏肺孢子虫全细胞裂解物进行了蛋白质印迹，并分析了免疫抑制后8-10周获得的大鼠血清和对照大鼠血清。无大鼠显示阳性抗体应答。这些老鼠没有被感染的原因尚不清楚。最近，我们从其他研究者那里获得了活的卡氏肺孢子虫，并对5只大鼠进行了气管接种。目前还没有确定他们中是否有人被感染。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 1) Plasmid construction and expression of recombinant proteins. To compare the antigenicity of our new p55 protein (p55-v3) with previously described p55 protein (p55-v0), we obtained the p55-v0 plasmid construct from Dr. Smulian, University of Cincinnati. Unexpectedly, we found that this construct did not express recombinant protein. DNA sequence analysis of multiple clones revealed a deletion of a single nucleotide A in the middle of the coding region, resulting in reading frame changes and introduction of multiple stop codons downstream. Therefore, we made new constructs by ourselves, with one construct for the full-length p55-v0 sequence and additional constructs for three overlapping fragments, which are corresponding to the fragments of p55-v3 we had expressed before. 2) Synthesis of peptides and production of antisera: We have obtained synthetic peptides and antisera, one specific for p55-v0 and two specific for p55-v3, from Sigma. The antisera will be used to study the epitope mapping and differential expression patterns of p55-v3 and p55-v0 as proposed in Specific Aim 1. 3) Animal models: We used 40 rats divided into 3 groups. Group A (10 rats) was a control group and housed in SPF conditions and did not receive any medication. Groups B (20 rats) and C (10 rats) were cohoused together in a conventional room (open cages without filter tops). Group B received immunosuppression with Depo-medrol to provoke spontaneous P. carinii pneumoniae (PCP). The dose of Depo-Medrol used was 2 mg/100g body weight subcutaneously twice a week for the first 4 weeks, and once a week for another 6 weeks. Group C didnt receive immunosuppression but were expected to have natural exposure to P. carinii from group B. After 3-4 weeks of immunosuppression all rats in Group B became sick and had apparent weight loss. Five rats died 8-10 weeks after immunosuppression. Examination of the lungs of these 5 rats by staining and PCR found that none of them were positive for P. carinii. In addition we did western blot with whole P. carinii cell lysates and analyzed sera from rats obtained at 8-10 weeks after immunosuppression and sera from control rats. No rats showed positive antibody responses. The reasons why these rats were not infected are unknown. Very recently we obtained live P. carinii organisms from other investigators and did transtracheal inoculation with 5 rats. It has not been determined if any of them are infected.